Analytical device for the automated determination of analytes in fluids

ABSTRACT

A method for determining the presence of an analyte in a fluid is described along with various components of an apparatus specifically designed to carry out the method. The method involves taking a reflectance reading from one surface of an inert porous matrix impregnated with a reagent that will interact with the analyte to produce a light-absorbing reaction product when the fluid being analyzed is applied to another surface and migrates through the matrix to the surface being read. Reflectance measurements are made at two separate wavelengths in order to eliminate interferences, and a timing circuit is triggered by an initial decrease in reflectance by the wetting of the surface whose reflectance is being measured by the fluid which passes through the inert matrix. The method and apparatus are particularly suitable for the measurement of glucose levels in blood without requiring separation of red blood cells from serum or plasma.

This is a division of application Ser. No. 07/896,418, filed Aug. 13,1986, now U.S. Pat. No. 4,935,346.

FIELD OF THE INVENTION

The present invention relates to a test device and method for thecolorimetric determination of chemical and biochemical components(analytes) in aqueous fluids, particularly whole blood. In one preferredembodiment it concerns a test device and method for colorimetricallymeasuring the concentration of glucose in whole blood.

BACKGROUND OF THE INVENTION

The quantification of chemical and biochemical components in coloredaqueous fluids, in particular colored biological fluids such as wholeblood and urine and biological fluid derivatives such as serum andplasma, is of ever-increasing importance. Important applications existin medical diagnosis and treatment and in the quantification of exposureto therapeutic drugs, intoxicants, hazardous chemicals and the like. Insome instances, the amounts of materials being determined are either sominiscule--in the range of a microgram or less per deciliter--or sodifficult to precisely determine that the apparatus employed iscomplicated and useful only to skilled laboratory personnel. In thiscase the results are generally not available for some hours or daysafter sampling. In other instances, there is often an emphasis on theability of lay operators to perform the test routinely, quickly andreproducibly outside a laboratory setting with rapid or immediateinformation display.

One common medical test is the measurement of blood glucose levels bydiabetics. Current teaching counsels diabetic patients to measure theirblood glucose level from two to seven times a day depending on thenature and severity of their individual cases. Based on the observedpattern in the measured glucose levels the patient and physiciantogether make adjustments in diet, exercise and insulin intake to bettermanage the disease. Clearly, this information should be available to thepatient immediately.

Currently a method widely used in the United States employs a testarticle of the type described in U.S. Pat. No. 3,298,789 issued Jan. 17,1967 to Mast. In this method a sample of fresh, whole blood (typically20-40 μl) is placed on an ethylcellulose-coated reagent pad containingan enzyme system having glucose oxidase and peroxidase activity. Theenzyme system reacts with glucose and releases hydrogen peroxide. Thepad also contains an indicator which reacts with the hydrogen peroxidein the presence of peroxidase to give a color proportional in intensityto the sample's glucose level.

Another popular blood glucose test method employs similar chemistry butin place of the ethylcellulose-coated pad employs a water-resistant filmthrough which the enzymes and indicator are dispersed. This type ofsystem is disclosed in U.S. Pat. No. 3,630,957 issued Dec. 28, 1971 toRey et al.

In both cases the sample is allowed to remain in contact with thereagent pad for a specified time (typically one minute). Then in thefirst case the blood sample is washed off with a stream of water whilein the second case it is wiped off the film. The reagent pad or film isthen blotted dry and evaluated. The evaluation is made either bycomparing color generated with a color chart or by placing the pad orfilm in a diffuse reflectance instrument to read a color intensityvalue.

While the above methods have been used in glucose monitoring for years,they do have certain limitations. The sample size required is ratherlarge for a finger stick test and is difficult to achieve for somepeople whose capillary blood does not express readily.

In addition, these methods share a limitation with other simplelay-operator colorimetric determinations in that their result is basedon an absolute color reading which is in turn related to the absoluteextent of reaction between the sample and the test reagents. The factthat the sample must be washed or wiped off the reagent pad after thetimed reaction interval requires that the user be ready at the end ofthe timed interval and wipe or apply a wash stream at the required time.The fact that the reaction is stopped by removing the sample leads tosome uncertainty in the result, especially in the hands of the homeuser. Overwashing can give low results and underwashing can give highresults.

Another problem that often exists in simple lay-operator colorimetricdeterminations is the necessity for initiating a timing sequence whenblood is applied to a reagent pad. A user will typically have conducteda finger stick to a obtain a blood sample and will then be required tosimultaneously apply the blood from the finger to a reagent pad whileinitiating a timing circuit with his or her other hand, therebyrequiring the use of both hands simultaneously. This is particularlydifficult since it is often necessary to insure that the timing circuitis started only when blood is applied to the reagent pad. All of theprior art methods require additional manipulations or additionalcircuitry to achieve this result. Accordingly, simplification of thisaspect of reflectance reading instruments is desirable.

The presence of red blood cells or other colored components ofteninterferes with the measurements of these absolute values therebycalling for exclusion of red blood cells in these two prior methods asthey are most widely practiced. In the device of U.S. Pat. No. 3,298,789an ethyl cellulose membrane prevents red blood cells from entering thereagent pad. Similarly, the water-resistant film of U.S. Pat. No.3,630,957 prevents red blood cells from entering. In both cases therinse or wipe also acts to remove these potentially interfering redblood cells prior to measurement.

Accordingly, there remains a need for a system of detecting analytes incolored liquids, such as blood, that does not require removal of excessliquid from a reflectance strip from which a reflectance reading isbeing obtained.

SUMMARY OF THE INVENTION

Novel methods, compositions and apparatus are provided for diagnosticassays comprising a hydrophilic porous matrix containing a signalproducing system and a reflectance measuring apparatus which isactivated upon a change in reflectance of the matrix when fluidpenetrates the matrix. The method comprises adding the sample, typicallywhole blood, to the matrix which filters out large particles, such asred blood cells, typically with the matrix present in the apparatus. Thesignal-producing system produces a product which further changes thereflectance of the matrix, which change can be related to the presenceof an analyte in a sample.

Exemplary of the diagnostic assay system is the determination of glucosein the whole blood, where the determination is made without interferencefrom the blood and without a complicated protocol subject to use error.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention can be more readily understood by reference to thefollowing detailed description when read in conjunction with theattached drawings, wherein:

FIG. 1 is a perspective view of one embodiment of a test devicecontaining the reaction pad to which the fluid being analyzed isapplied.

FIG. 2 is a block diagram schematic of an apparatus that can be employedin the practice of the invention.

FIG. 3 is a block diagram schematic of an alternate apparatus that canbe employed in the practice of the invention.

DETAILED DESCRIPTION OF THE INVENTION The Reagent Element

The subject invention provides an improved rapid and simple methodologyemploying reliable and easy to operate apparatus for the determinationof analytes such as glucose, particularly involving an enzyme substratewhich results in the production of hydrogen peroxide as an enzymeproduct. The method involves applying to a porous matrix a small volumeof whole blood, sufficient to saturate the matrix. Bound to the matrixare one or more reagents of a signal producing system, which results inthe production of a product resulting an initial change in the amount ofreflectance of the matrix. The matrix is typically present in areflectance-measuring apparatus when blood is applied. The liquid samplepenetrates the matrix, resulting in an initial change in reflectance atthe measurement surface. A reading is then taken at one or more timesafter the initial change in reflectance to relate the further change inreflectance at the measurement surface or in the matrix as a result offormation of the reaction product to the amount of analyte in thesample.

For measurements in blood, particularly glucose measurements, wholeblood is typically used as the assay medium. The matrix contains anoxidase enzyme which produces hydrogen peroxide. Also contained in thematrix will be a second enzyme, particularly a peroxidase, and a dyesystem which produces a light-absorbing product in conjunction with theperoxidase. The light-absorbing product changes the reflectance signal.With whole blood, readings are taken at two different wavelengths withthe reading at one wavelength used to subtract out backgroundinterference caused by hematocrit, blood oxygenation, and othervariables which may affect the result.

A reagent element is employed which comprises the matrix and the membersof the signal producing system contained within the matrix. The reagentelement may include other components for particular applications. Themethod requires applying a small volume of blood, which typically hasnot been subject to prior treatment (other than optional treatment withan anticoagulant), to the matrix. Timing of the measurement is activatedby the apparatus automatically detecting a change in reflectance of thematrix when fluid penetrates the matrix. The change in reflectance overa predetermined time period as a result of formation of reaction productis then related to the amount of analyte in a sample.

The first component of the present invention to be considered is areagent element, conveniently in the shape of a pad, comprising an inertporous matrix and the component or components of a signal-producingsystem, which system is capable of reacting with an analyte to produce alight-absorbing reaction product, impregnated into the pores of theporous matrix. The signal-producing system does not significantly impedethe flow of liquid through the matrix.

In order to assist in reading reflectance, it is preferred that thematrix have at least one side which is substantially smooth and flat.Typically, the matrix will be formed into a thin sheet with at least onesmooth, flat side. In use, the liquid sample being analyzed is appliedto one side of the sheet whereby any assay compound present passesthrough the reagent element by means of capillary, wicking, gravity flowand/or diffusion actions. The components of the signal producing systempresent in the matrix will react to give a light absorbing reactionproduct. Incident light impinges upon the reagent element at a locationother than the location to which the sample is applied. Light isreflected from the surface of the element as diffuse reflected light.This diffuse light is collected and measured, for example by thedetector of a reflectance spectrophotometer. The amount of reflectedlight will be related to the amount of analyte in the sample, usuallybeing an inverse function of the amount of analyte in the sample.

The Matrix

Each of the components necessary for producing the reagent element willbe described in turn. The first component is the matrix itself.

The matrix will be a hydrophilic porous matrix to which reagents may becovalently or non-covalently bound. The matrix will allow for the flowof an aqueous medium through the matrix. It will also allow for bindingof protein compositions to the matrix without significantly adverselyaffecting the biological activity of the protein, e.g. enzymaticactivity of an enzyme. To the extent that proteins are to be covalentlybound, the matrix will have active sites for covalent bonding or may beactivated by means known to the art. The composition of the matrix willbe reflective and will be of sufficient thickness to permit theformation of a light-absorbing dye in the void volume or on the surfaceto substantially affect the reflectance from the matrix. The matrix maybe of a uniform composition or a coating on a substrate providing thenecessary structure and physical properties.

The matrix will usually not deform on wetting, thus retaining itsoriginal conformation and size. The matrix will have a definedabsorbtivity, so that the volume which is absorbed can be calibratedwithin reasonable limits, variations usually being maintained belowabout 50%, preferably not greater than 10%. The matrix will havesufficient wet strength to allow for routine manufacture. The matrixwill permit non-covalently bound reagents to be relatively uniformlydistributed on the surface of the matrix.

As exemplary of matrix surfaces are polyamides, particularly withsamples involving whole blood. The polyamides are convenientlycondensation polymers of monomers of from 4 to 8 carbon atoms, where themonomers are lactams or combinations of diamines and di-carboxylicacids. Other polymeric compositions having comparable properties mayalso find use. The polyamide compositions may be modified to introduceother functional groups which provide for charged structures, so thatthe surfaces of the matrix may be neutral, positive or negative, as wellas neutral, basic or acidic. Preferred surfaces are positively charged.

When used with whole blood, the porous matrix preferably has pores withan average diameter in the range of from about 0.1 to 2.0 μm, morepreferrably from about 0.6 to 1.0 μm.

A preferred manner of preparing the porous material is to cast thehydrophilic polymer onto a core of non-woven fibers. The core fibers canbe any fibrous material that produce the described integrity andstrength, such as polyesters and polyamides. The reagent that will formthe light-absorbing reaction product, which is discussed later indetail, is present within the pores of the matrix but does not block thematrix so that the liquid portion of the assay medium, e.g. blood, beinganalyzed can flow through the pores of the matrix, while particles, suchas erythrocytes, are held at the surface.

The matrix is substantially reflective so that it gives a diffusereflectance without the use of a reflective backing. Preferably at least25%, more preferably at least 50%, of the incident light applied to thematrix is reflected and emitted as diffuse reflectance. A matrix of lessthan about 0.5 mm thickness is usually employed, with from about 0.01 to0.3 mm being preferred. A thickness of from 0.1 to 0.2 mm is mostpreferred, particularly for a nylon matrix.

Typically, the matrix will be attached to a holder in order to give itphysical form and rigidity, although this may not be necessary. FIG. 1shows one embodiment of the invention in which a thin hydrophilic matrixpad 11 is positioned at one end of a plastic holder 12 by means of anadhesive 13 which directly and firmly attaches the reagent pad to thehandle. A hole 14 is present in the plastic holder 12 in the area towhich reagent pad 11 is attached so that sample can be applied to oneside of the reagent pad and light reflected from the other side.

A liquid sample to be tested is applied to pad 11. Generally, with bloodbeing exemplary of a sample being tested, the reagent pad will be on theorder of about 10 mm² to 100 mm² in surface area, especially 10 mm² to50 mm² in area, which is normally a volume that 5-10 microliters ofsample will more than saturate.

Diffuse reflectance measurements in the prior art have typically beentaken using a reflective backing attached to or placed behind thematrix. No such backing is needed or will normally be present during thepractice of the present invention, either as part of the reagent elementor the reflectance apparatus.

As can be seen from FIG. 1, the support holds reagent pad 11 so that asample can be applied to one side of the reagent pad while lightreflectance is measured from the side of the reagent pad opposite thelocation where sample is applied.

FIG. 2 shows a system in which the reagent is applied to the side withthe hole in the backing handle while light is reflected and measured onthe other side of the reagent pad. Other structures than the onedepicted may be employed. The pad may take various shapes and forms,subject to the limitations provided herein. The pad will be accessibleon at least one surface and usually two surfaces.

The hydrophilic layer (reagent element) may be attached to the supportby any convenient means, e.g., a holder, clamp or adhesives; however, inthe preferred method it is bonded to the backing. The bonding can bedone with any non-reactive adhesive, by a thermal method in which thebacking surface is melted enough to entrap some of the material used forthe hydrophilic layer, or by microwave or ultrasonic bonding methodswhich likewise fuse the hydrophilic sample pads to the backing. It isimportant that the bonding be such as to not itself interferesubstantially with the diffuse reflectance measurements or the reactionbeing measured, although this is unlikely to occur as no adhesive needbe present at the location where the reading is taken. For example, anadhesive 13 can be applied to the backing strip 12 followed first bypunching hole 14 into the combined strip and adhesive and then applyingreagent pad 11 to the adhesive in the vicinity of hole 14 so that theperipheral portion of the reagent pad attaches to the backing strip.

The Chemical Reagents

Any signal producing system may be employed that is capable of reactingwith the analyte in the sample to produce (either directly orindirectly) a compound that is characteristically absorptive at awavelength other than a wavelength at which the assay mediumsubstantially absorbs.

Polyamide matrices are particularly useful for carrying out reactions inwhich a substrate (analyte) reacts with an oxygen-utilizing oxidaseenzyme in such a manner that a product is produced that further reactswith a dye intermediate to either directly or indirectly form a dyewhich absorbs in a predetermined wavelength range. For example, anoxidase enzyme can oxidize a substrate and produce hydrogen peroxide asa reaction product. The hydrogen peroxide can then react with a dyeintermediate or precursor, in a catalysed or uncatalysed reaction, toproduce an oxidized form of the intermediate or precursor. This oxidizedmaterial may produce the colored product or react with a secondprecursor to form the final dye.

Nonlimiting examples of analyses and typical reagents include thefollowing materials shown in the following list.

    ______________________________________                                        Analyte and Sample Type                                                                         Reagents                                                    ______________________________________                                        Glucose in blood, serum,                                                                        Glucose Oxidase, Peroxi-                                    urine or other biological                                                                       dase and an Oxygen                                          fluids, wine, fruit juices                                                                      Acceptor                                                    or other colored aqueous                                                      fluids. Whole blood is a                                                                        Oxygen Acceptors include:                                   particularly preferred                                                                          O-dianisidine (1)                                           sample type.      O-toluidine                                                                   O-tolidine (1)                                                                Benzidine (1)                                                                 2,2'-Azinodi-(3-ethylbenz-                                                    thiazoline sulphonic                                                          acid-(6)) (1)                                                                 3-Methyl-2-benzothiazoli-                                                     none hydrazone plus N,N-                                                      dimethylaniline (1)                                                           Phenol plus 4-aminophena-                                                     zone (1)                                                                      Sulfonated 2,4-dichloro-                                                      phenol plus 4-amino-                                                          phenazone (2)                                                                 3-Methyl-2-benzothiazoli-                                                     none hydrazone plus 3-                                                        (dimethylamino)benzoic                                                        acid (3)                                                                      2-Methoxy-4-allyl phenol (4)                                                  4-Aminoantipyrine-                                                            dimethylaniline (5)                                         ______________________________________                                         (1) As reported Clinical Chemistry, Richterich and Columbo, p. 367 and        references cited therein.                                                     (2) Analyst, 97, (1972) 142-5.                                                (3) Anal. Biochem., 105, (1980) 389-397.                                      (4) Anal. Biochem., 79, (1977) 597-601.                                       (5) Clinica Chemica Acta, 75, (1977) 387-391 all incorporated herein by       reference.                                                               

The Analysis Method

The analysis method of this invention relies on a change in absorbance,as measured by diffuse reflectance, which is dependent upon the amountof analyte present in a sample being tested. This change may bedetermined by measuring the change in the absorbance of the test samplebetween two or more points in time.

The first step of the assay to be considered will be application of thesample to the matrix. In practice, an analysis could be carried out asfollows: First a sample of aqueous fluid containing an analyte isobtained. Blood may be obtained by a finger stick, for example. Anexcess over matrix saturation in the area where reflectance will bemeasured (i.e., about 5-10 microliters) of this fluid is applied to thereagent element or elements of the test device. Simultaneous starting ofa timer is not required (as is commonly required in the prior art), aswill become clear below. Excess fluid can be removed, such as by lightblotting, but such removal is also not required. The test device istypically mounted in an instrument for reading light absorbance; e.g.,color intensity, by reflectance, prior to application of the sample.Absorbance is measured at certain points in time after application ofthe sample. Absorbance refers in this application not only to lightwithin the visual wavelength range but also outside the visualwavelength range, such as infrared and ultraviolet radiation. From thesemeasurements of absorbance a rate of color development can be calibratedin terms of analyte level.

The Measuring Instrument

A suitable instrument, such as a diffuse reflectance spectrophotometerwith appropriate software, can be made to automatically read reflectanceat certain points in time, calculate rate of reflectance change, and,using calibration factors, output the level of analyte in the aqueousfluid. Such a device is schematically shown in FIG. 2 wherein a testdevice of the invention comprising backing 12 to which reagent pad 11 isaffixed is shown. Light source 14,14', for example a high intensitylight emitting diode (LED), projects a beam of light onto the reagentpad. A substantial portion (at least 25%, preferably at least 35%, andmore preferably at least 50%, in the absence of reaction product) ofthis light is diffusively reflected from the reagent pad and is detectedby light detector 15, for example a phototransistor that produces anoutput current proportional to the light it receives. Light source14,14' and/or detector 15 can be adapted to generate or respond to aparticular wavelength light, if desired. The output of detector 15 ispassed to amplifier 16, for example, a linear integrated circuit whichconverts the phototransistor current to a voltage. The output ofamplifier 16 can be fed to track and hold circuit 17. This is acombination linear/digital integrated circuit which tracks or followsthe analog voltage from amplifier 16 and, upon command frommicroprocessor 20, locks or holds the voltage at its level at that time.Analog-to-digital converter 19 takes the analog voltage from track andhold circuit 17 and converts it to, for example, a twelve-bit binarydigital number upon command of microprocessor 20. Microprocessor 20 canbe a digital integrated circuit. It serves the following controlfunctions: 1) timing for the entire system; 2) reading of the output ofanalog/digital converter 19; 3) together with program and data memory21, storing data corresponding to the reflectance measured at specifiedtime intervals; 4) calculating analyte levels from the storedreflectances; and 5) outputing analyte concentration data to display 22.Memory 21 can be a digital integrated circuit which stores data and themicroprocessor operating program. Display 22 can take various hard copyand soft copy forms. Usually it is a visual display, such as a liquidcrystal or LED display, but it can also be a tape printer, audiblesignal, or the like. The instrument also can include a start-stop switchand can provide an audible or visible time output to indicate times forapplying samples, taking readings, etc., if desired.

Reflectance Switching

In the present invention, the reflectance circuit itself can be used toinitiate timing by measuring a drop in reflectance that occurs when theaqueous portion of the suspension solution applied to the reagent pad(e.g., blood) migrates to the surface at which reflectance is beingmeasured. Typically, the measuring device is turned on in a "ready" modein which reflectance readings are automatically made at closely spacedintervals (typically about 0.2 seconds) from the typically off-white,substantially dry, unreacted reagent strip. The initial measurement istypically made prior to penetration of the matrix by fluid beinganalyzed but can be made after the fluid has been applied to a locationon the reagent element other than where reflectance is being measured.The reflectance value is evaluated by the microprocessor, typically bystoring successive values in memory and then comparing each value withthe initial unreacted value. When the aqueous solution penetrates thereagent matrix, the drop in reflectance signals the start of themeasuring time interval. Drops in reflectance of 5-50% can be used toinitiate timing, typically a drop of about 10%. In this simple way thereis exact synchronization of assay medium reaching the surface from whichmeasurements are taken and initiation of the sequence of readings, withno requirement of activity by the user.

Although the total systems described in this application areparticularly directed to the use of polyamide matrices and particularlyto the use of such matrices in determining the concentration of varioussugars, such as glucose, and other materials of biological origin, thereis no need to limit the reflectance switching aspect of the invention tosuch matrices. For example, the matrix used with reflectance switchingmay be formed from any water-insoluble hydrophilic material and anyother type of reflectance assay.

Particular Application to Glucose Assay

A particular example with regard to detecting glucose in the presence ofred blood cells will now be given in order that greater detail andparticular advantage can be pointed out. Although this represents apreferred embodiment of the present invention, the invention is notlimited to the detection of glucose in blood.

The use of polyamide surfaces to form the reagent element provides anumber of desirable characteristics in the present invention. These arethat the reagent element is hydrophilic (i.e., takes up reagent andsample readily), does not deform on wetting (so as to provide a flatsurface for reflectance reading), is compatible with enzymes (in orderto impart good shelf stability), takes up a limited sample volume perunit volume of membrane (necessary in order to demonstrate an extendeddynamic range of measurements), and shows sufficient wet strength toallow for routine manufacture.

In a typical configuration, the method is carried out using an apparatusconsisting of a plastic holder and the reagent element (the matrixhaving the signal producing system impregnated therein). The preferredmatrix for use in preparing the reagent element is a nylonmicrofiltration membrane, particularly membranes made from nylon-66 caston a core of non-woven polyester fibers. Numerous nylon microfiltrationmembranes of this class are produced commercially by the Pall UltrafineFiltration Corporation having average pore sizes from 0.1 to 3.0microns. These materials show mechanical strength and flexibility,dimensional stability upon exposure to water, and rapid wetting.

Many variations in specific chemical structure of the nylon arepossible. These include unfunctionalized nylon-66 with charged endgroups (sold under the trademark ULTIPORE by Pall Ultrafine FiltrationCorporation; "Pall"). Positive charges predominate below pH 6 whilenegative charges predominate above pH 6. In other membranes the nylon isfunctionalized before the membrane is formed to give membranes withdifferent properties. Nylons functionalized with carboxy groups arenegatively charged over a wide pH range (sold as CARBOXYDYNE by Pall).Nylons can also be functionalized with a high density of positivelycharged groups on its surface, typically quaternary amine groups, sothat they display little variation in charge over a wide pH range (soldas POSIDYNE by Pall). Such materials are particularly well suited forthe practice of the present invention. It is also possible to usemembranes having reactive functional groups designed for covalentimmobilization of proteins (sold as BIODYNE Immuno Affinity membranes byPall). Such materials can be used to covalently attach proteins, e.g.enzymes, used as reagents. Although all of these materials are usable,nylon having a high density of positively charged groups on its surfaceprovide the best stability of reagents when formulated into a dryreagent pad. Unfunctionalized nylon gives the next best stability withthe carboxylated nylons next best.

Desirable results can be obtained with pore sizes ranging from about0.2-2.0 μm, preferably about 0.5-1.2 μm, and most preferably about 0.8μm, when used with whole blood.

The form of the handle on which the reagent element is assembled isrelatively unimportant as long as the handle allows access to one sideof the reagent element by sample and to the other side of the reagentelement by incident light whose reflectance is being measured. Thehandle also aids in inserting the reagent element into the absorbancemeasuring device so that it registers with the optical system. Oneexample of a suitable handle is a mylar or other plastic strip to whicha transfer adhesive such as 3M 465 or Y9460 transfer adhesive has beenapplied. A hole is punched into the plastic through the transferadhesive. A reagent element, typically in the form of a thin pad, eithercontaining reagents or to which reagents will later be added, is thenapplied to the handle by means of the transfer adhesive so that it isfirmly attached to the handle in the area surrounding the hole that hasbeen punched through the handle and the transfer adhesive. Such a deviceis illustrated in FIG. 1, which shows reagent pad 11 attached to a Mylarhandle 12 by means of adhesive 13. Hole 14 allows access of the sampleor incident light to one side of reagent pad 11 while access to theother side of the reagent pad is unrestricted. All dimensions of thereagent pad and handle can be selected so that the reagent pad fitssecurely into a reflectance-reading instrument in proximal location to alight source and a reflected-light detector.

If a nylon matrix is selected to form the reagent pad, when theindicated thicknesses are employed, it is preferred to have the reagentpad supported by the holder in such a manner that no more than 6 mm,measured in any direction, is unsupported by the holder at the locationwere the sample is applied and light reflectance is measured. Largerunsupported areas tend to provide inadequate dimensional stability tothe membrane so that measurement of reflectance from the surface isadversely affected. A 5 mm diameter hole 14 in the reagent strip shownin FIG. 1 works quite satisfactorily.

There is no particular limit on the minimum diameter of such a hole,although diameters of at least 2 mm are preferred for ease ofmanufacture, sample application, and light reflectance reading.

Although a number of dyes could be used as indicators, the choice willdepend upon the nature of the sample. It is necessary to select a dyehaving an absorbance at a wavelength different from the wavelength atwhich red blood cells absorb light, with whole blood as the assaymedium, or other contaminants in the solution being analyzed with otherassay media. The MBTH-DMAB dye couple (3-methyl-2-benzothiazolinonehydrazone hydrochloride and 3-dimethylaminobenzoic acid), although beingpreviously described as suitable for color development for peroxidaselabels in enzyme immunoassays, has never been used in a commercialglucose measuring reagent. This dye couple gives greater dynamic rangeand shows improved enzymatic stability as compared to traditional dyesused for glucose measurement, such as benzidine derivatives.Furthermore, the MBTH-DMAB dye couple is not carcinogenic, acharacteristic of most benzidine derivatives.

Another dye couple that can be used in the measurement of glucose is theAAP-CTA (4-aminoantipyrene and chromotropic acid) couple. Although thiscouple does not provide as broad a dynamic range as MBTH-DMAB, it isstable and suitable for use in the practice of the present inventionwhen measuring glucose. Again, the AAP-CTA dye couple provides anexpanded dynamic range and greater enzymatic activity stability than themore widely used benzidine dyes.

The use of the MBTH-DMAB couple allows for correction of hematocrit anddegree of oxygenation of blood with a single correction factor. The moretypically used benzidine dyes do not permit such a correction. The dyeforms a chromophore that absorbs at approximately 635 nm but not to anysignificant extent at 700 nm. Slight variations in measuring wavelengths(±about 10 nm) are permitted. At 700 nm both hematocrit and degree ofoxygenation can be measured by measuring blood color. Furthermore, lightemitting diodes (LED) are commercially available for both 635 nm and 700nm measurements, thereby simplifying mass-production of a device. Byusing the preferred membrane pore size described above and the subjectreagent formulation both hematocrit and oxygenation behavior can becorrected by measuring at the single 700 nm wavelength.

Two additional conditions were found to provide particular stability andlong shelf life for a glucose oxidase/peroxidase formulation on apolyamide matrix. These use a pH in the range of 3.8 to 5.0, preferablyabout 3.8 to 4.3, most preferably about 4.0, and use of a concentratedbuffer system for applying the reagents to the matrix. The mosteffective buffer was found to be 10 weight percent citrate buffer, withconcentrations of from 5-15% being effective. These are weight/volumepercentages of the solution in which the reagents are applied to thematrix. Other buffers can be used on the same molar basis. Greateststability was achieved using a low pH, preferably about pH 4, anMBTH-DMAB dye system, and a high enzyme concentration of approximately500-1000 U/ml of application solution.

In preparing the MBTH-DMAB reagent and the enzyme system that forms theremainder of the signal producing system, it is not necessary tomaintain exact volumes and ratios although the suggested values belowgive good results. Reagents are readily absorbed by the matrix pad whenthe glucose oxidase is present in a solution at about 27-54% by volume,the peroxidase is present at a concentration of about 2.7-5.4 mg/ml,MBTH is present at a concentration of about 4-8 mg/ml, and DMAB ispresent at a concentration of about 8-16 mg/ml. The DMAB-MBTH weightratio is preferably maintained in the range of (1-4):1, preferably about(1.5-2.5):1, most preferably about 2:1.

The basic manufacturing techniques for the reagent element are, onceestablished, straightforward. The membrane itself is strong and stable,particularly when a nylon membrane of the preferred embodiment isselected. Only two solutions are necessary for applying reagent, andthese solutions are both readily formulated and stable. The firstgenerally contains the dye components and the second generally containsthe enzymes. When using the MBTH-DMAB dye couple, for example, theindividual dyes are dissolved in an aqueous organic solvent, typically a1:1 mixture of acetonitrile and water. The matrix is dipped into thesolution, excess liquid is removed by blotting, and the matrix is thendried, typically at 50°-60° C. for 10-20 minutes. The matrix containingthe dyes is then dipped into an aqueous solution containing the enzymes.A typical formulation would contain the peroxidase and glucose oxidaseenzymes as well as any desired buffer, preservative, stabilizer, or thelike. The matrix is then blotted to remove excess liquid and dried asbefore. A typical formulation for the glucose reagent is as follows:

Dye dip

Combine:

40 mg MBTH,

80 mg DMAB,

5 ml acetonitrile, and

5 ml water.

Stir until all solids are dissolved and pour onto a glass plate or otherflat surface. Dip a piece of posidyne membrane (Pall Co.), blot offexcess liquid, and dry at 56° C. for 15 minutes.

Enzyme dip

Combine:

6 ml water,

10 mg EDTA, disodium salt,

200 mg Poly Pep, low viscosity, (Sigma),

0.668 g sodium citrate,

0.523 g citric acid,

2.0 ml 6 wt % Gantrez AN-139 dissolved in water (GAF)

30 mg horseradish peroxidase, 100 units/mg, and

3.0 ml glucose oxidase, 2000 units/ml.

Stir until all solids are dissolved and pour onto a glass plate or otherflat surface. Dip a piece of membrane previously impregnated with dyes,blot off excess liquid, and dry at 56° C. for 15 minutes.

The electronic apparatus used to make the reflectance readings minimallycontains a light source, a reflected light detector, an amplifier, ananalog to digital converter, a microprocessor with memory and program,and a display device.

The light source typically consists of a light emitting diode (LED).Although it is possible to use a polychromic light source and a lightdetector capable of measuring at two different wavelengths, a preferredapparatus would contain two LED sources or a single diode capable ofemitting two distinct wavelengths of light. Commercially available LEDsproducing the wavelengths of light described as being preferred in thepresent specification include a Hewlett Packard HLMP-1340 with anemission maximum at 635 nm and a Hewlett Packard QEMT-1045 with anarrow-band emission maximum at 700 nm. Suitable commercially availablelight detectors include a Hammamatsu 5874-18K and a Litronix BPX-65.

Although other methods of taking measurements are feasible, thefollowing method has provided the desired results. Readings are taken bythe photodetector at specified intervals after timing is initiated. The635 nm LED is powered only during a brief measuring time span thatbegins approximately 20 seconds after the start time as indicated byreflectance switching. If this reading indicates that a high level ofglucose is present in the sample, a 30-second reading is taken and usedin the final calculation in order to improve accuracy. Typically, highlevels are considered to begin at about 250 mg/dl. The background iscorrected with a 700 nm reading taken about 15 seconds after the startof the measurement period. The reading from the photodetector isintegrated over the interval while the appropriate LED is activated,which is typically less than one second. The raw reflectance readingsare then used for calculations performed by the microprocessor after thesignal has been amplified and converted to a digital signal. Numerousmicroprocessors can be used to carry out the calculation. An AIM65single-board microcomputer manufactured by Rockwell International hasproven to be satisfactory.

The present methods and apparatuses allow a very simple procedure withminimum operational steps on the part of the user. In use, the reagentstrip is placed in the detector so that the hole in the strip registerswith the optics of the detecting system. A removable cap or other coveris placed over the optics and strip to shield the assembly from ambientlight. The measurement sequence is then initiated by pressing a buttonon the measuring apparatus that activates the microcomputer to take ameasurement of reflected light from the unreacted reagent pad, called anR_(dry) reading. The cap is then removed and a drop of blood is appliedto the reagent pad, typically while the reagent pad is registered withthe optics and the reading device. It is preferred that the reagentstrip be left in register with the optics in order to minimize handling.The instrument is capable of sensing the application of blood or othersample by a decrease in the reflectance when the sample passes throughthe matrix and reflected light is measured on the opposite side. Thedecrease in reflectance initiates a timing sequence which is describedin detail at other locations in this specification. The cover should bereplaced within 15 seconds of sample application, although this time mayvary depending on the type of sample being measured. Results aretypically displayed at approximately 30 seconds after blood applicationwhen a blood glucose sample is being measured, although a 20 secondreaction is permissible for glucose samples having a concentration ofglucose of less than 250 mg/dl. If other samples are being measured,suitable times for displaying the result may differ and can be readilydetermined from the characteristics of the reagent/sample selected.

A particularly accurate evaluation of glucose level (or any otheranalyte being measured) can be made using the background current, i.e.,the current from the photodetector with power on but with no lightreflected from the reagent pad, in order to make a backgroundcorrection. It has been demonstrated that over a 2-3 month period thatthis value does not change for a particular instrument preparedaccording to the preferred embodiments of this specification, and it ispossible to program this background reading into the computer memory asa constant. With a slight modification of the procedure, however, thisvalue can be measured with each analysis for more accurate results. Inthe modified procedure the meter would be turned on with the lid closedbefore the reagent strip is in place, and the background current wouldbe measured. The test strip would then be inserted into the meter withthe cover closed, an R_(dry) measurement taken, and the procedurecontinued as described above. With this modified procedure thebackground current does not need to be stable throughout the life of themeter, thereby providing more accurate results.

The raw data necessary for calculating a result in a glucose assay are abackground current reported as background reflectance, R_(b), asdescribed above; a reading of the unreacted test strip, R_(dry), alsodescribed above; and an endpoint measurement. Using the preferredembodiments described herein, the endpoint is not particularly stableand must be precisely timed from the initial application of blood.However, the meter as described herein performs this timingautomatically. For glucose concentrations below 250 mg/dl, a suitablystable endpoint is reached in 20 seconds, and a final reflectance, R₂₀,is taken. For glucose concentrations up to 450 mg/dl, a 30-secondreflectance reading, R₃₀, is adequate. Although the system describedherein displays good differentiation up to 800 mg/dl of glucose, themeasurement is somewhat noisy and inaccurate above 450 mg/dl, althoughnot so great as to cause a significant problem. Longer reaction timesshould provide more suitable readings for the higher levels of glucoseconcentration.

The 700 nm reflectance reading for the dual wavelength measurement istypically taken at 15 seconds (R₁₅). By this time blood will havecompletely saturated the reagent pad. Beyond 15 seconds the dye reactioncontinues to take place and is sensed, to a small part, by a 700 nmreading. Accordingly, since dye absorption by the 700 nm signal is adisadvantage, readings beyond 15 seconds are ignored in thecalculations.

The raw data described above are used to calculate parametersproportional to glucose concentration which can be more easilyvisualized than reflectance measurements. A logarithmic transformationof reflectance analogous to the relationship between absorbance andanalyte concentration observed in transmission spectroscopy (Beer's Law)can be used if desired. A simplification of the Kubelka-Monk equations,derived specifically for reflectance spectroscopy, have provenparticularly useful. In this derivation K/S is related to analyteconcentration with K/S defined by Equation 1.

    K/S-t=(1-R*t).sup.2 /(2×R*t)                         (1)

R*t is the reflectivity taken at a particular endpoint time, t, and isthe absorbed fraction of the incident light beam described by Equation2, where R_(t) is the endpoint reflectance, R₂₀ or R₃₀.

    R*t=(R.sub.t -R.sub.b)/(R.sub.dry -R.sub.b)                (2)

R*t varies from 0 for no reflected light (R_(b)) to 1 for totalreflected light (R_(dry)). The use of reflectivity in the calculationsgreatly simplifies meter design as a highly stable source and adetection circuit become unnecessary since these components aremonitored with each R_(dry) and R_(b) measurement.

For a single wavelength reading K/S can be calculated at 20 seconds(K/S-20) or 30 seconds (K/S-30). The calibration curves relating theseparameters to YSI (Yellow Springs Instruments) glucose measurements canbe precisely described by the third order polynomial equation outlinedin Equation 3.

    YSI=a.sub.0 +a.sub.1 (K/S)+a.sub.2 (K/S).sup.2 +a.sub.3 (K/S).sup.3(3)

The coefficients for these polynomials are listed in Table 1.

                  TABLE 1                                                         ______________________________________                                        Coefficients for Third Order Polynomial Fit of                                Single Wavelength Calibration Curves                                                  K/S-20        K/S-30                                                  ______________________________________                                        a.sub.0   -55.75          -55.25                                              a.sub.1   0.1632          0.1334                                              a.sub.2   -5.765 × 10.sup.-5                                                                      -2.241 × 10.sup.-5                            a.sub.3     2.58 × 10.sup.-8                                                                        1.20 × 10.sup.-8                            ______________________________________                                    

The single chemical species being measured in the preferred embodimentsis the MBTH-DMAB indamine dye and the complex matrix being analyzed iswhole blood distributed on a 0.8 μm POSIDYNE membrane. A review entitled"Application of Near Infra Red Spectrophotometry to the NondestructiveAnalysis of Foods: A Review of Experimental Results", CRC CriticalReviews in Food Science and Nutrition, 18(3) 203-30 (1983), describesthe use of instruments based on the measurement of an optical densitydifference ΔOD (λ_(a) -λ_(b)) where ODλ_(a) is the optical density ofthe wavelength corresponding to the absorption maximum of a component tobe determined and ODλ_(b) is the optical density at a wavelength wherethe same component does not absorb significantly.

The algorithm for dual wavelength measurement is by necessity morecomplex than for single wavelength measurement but is much morepowerful. The first order correction applied by the 700 nm reading is asimple subtraction of background color due to blood. In order to makethis correction, a relationship between absorbance at 635 nm and 700 nmdue to blood color can be and was determined by measuring blood sampleswith 0 mg/dl glucose over a wide range of blood color. The color rangewas constructed by varying hematocrit, and fairly linear relationshipswere observed. From these lines the K/S-15 at 700 nm was normalized togive equivalence to the K/S-30 at 635 nm. This relationship is reportedin Equation 4, where K/S-15 n is the normalized K/S-15 at 700 nm.

    K/S-15n=(K/S-15×1.54)-0.133                          (4)

Note that the equivalence of the normalized 700 nm signal and the 635 nmsignal is only true at zero glucose. The expressions from which thecalibration curves were derived are defined by Equations 5 and 6.

    K/S-20/15=(K/S-20)-(K/S-15n)                               (5)

    K/S-30/15=(K/S-30)-(K/S-15n)                               (6)

These curves are best fit by fourth-order polynomial equations similarto Equation 3 to which a fourth-order term in K/S is added. Thecomputer-fit coefficients for these equations are listed in Table 2.

                  TABLE 2                                                         ______________________________________                                        Coefficients for Fourth-Order Polynomial Fit of                               Dual Wavelength Calibration Curves                                                    K/S-20/15     K/S-30/15                                               ______________________________________                                        a.sub.0   -0.1388         1.099                                               a.sub.1   0.1064          0.05235                                             a.sub.2    6.259 × 10.sup.-5                                                                      1.229 × 10.sup.-4                             a.sub.3   -6.12 × 10.sup.-8                                                                       -5.83 × 10.sup.-8                             a.sub.4    .sup. 3.21 × 10.sup.-11                                                                 .sup. 1.30 × 10.sup.-11                      ______________________________________                                    

A second order correction to eliminate errors due to chromatographyeffects has also been developed. Low hematocrit samples havecharacteristically low 700 nm readings compared to higher hematocritsamples with the same 635 nm reading. When the ratio of(K/S-30)/(K/S-15) is plotted versus K/S-30 over a wide range ofhematocrits and glucose concentrations, the resulting line on the graphindicates the border between samples which display chromatographyeffects (above the curve) and those that do not (below the curve). TheK/S-30 for the samples above the curve are corrected by elevating thereading to correspond to a point on the curve with the same(K/S-30)/(K/S-15).

The correction factors reported above were tailor made to fit a singleinstrument and a limited number of reagent preparations. The algorithmcan be optimized for an individual instrument and reagent in the samemanner that is described above.

In summary, the system of the present invention minimizes operatoractions and provides numerous advantages over prior artreflectance-reading methods. When compared to prior methods fordetermining glucose in blood, for example, there are several apparentadvantages. First, the amount of sample required to saturate the thinreagent pad is small (typically 5-10 microliters). Second, operator timerequired is only that necessary to apply the sample to the thinhydrophilic layer and close the cover (typically 4-7 seconds). Third, nosimultaneous timing start is required. Fourth, whole blood can be used.The method does not require any separation or utilization ofred-cell-free samples and likewise can be used with other deeply coloredsamples.

Several unobvious advantages arise as a result of the practice of thepresent invention with whole blood. Normally, aqueous solutions (likeblood) will penetrate a hydrophilic membrane to give a liquid layer onthe opposite side of the membrane, a surface that is then not suited fora reflectance measurement. It has been discovered, however, that blood,apparently because of interactions of red blood cells and proteins inthe blood with the matrix, will wet the polyamide matrix without havingan excess liquid penetrate the porous matrix to interfere with thereflectance reading on the opposite side of the matrix.

Furthermore, the thin membranes used in the present invention would beexpected when wet to transmit light and return only a weak signal to thereflectance measuring device. Prior teachings have generally indicatedthat a reflective layer is necessary behind the matrix in order toreflect sufficient light. In other cases a white pad has been placedbehind the reagent pad prior to color measurement. In the present case,neither a reflective layer or a white pad is required. In fact, theinvention is typically carried out with a light-absorbing surface behindthe reagent element when incident light is impinged upon the matrix.Using a light-absorbing surface behind the reagent element, coupled withmeasuring reflectance at two different wavelengths, allows acceptablereflectance measurements to be obtained without removal of excess liquidfrom the matrix, thereby eliminating a step typically required byprevious teachings.

The invention now being generally described, the same will be betterunderstood by reference to the following specific examples which arepresented for purposes of illustration only and are not to be consideredlimiting of the invention unless so specified.

EXAMPLE I

In this and the following examples, "MPX" refers to the system of thepresent invention.

Reproducibility

One male blood sample (JG, hematocrit=45) was used to collect thereproducibility data set forth in Tables 3-5.

                  TABLE 3                                                         ______________________________________                                        Reproducibility of a Single Wavelength MPX System                             YSI     Average (mg/dl)                                                                            S.D. (mg/dl)                                                                              % C.V.                                       (mg/dl) 20 sec.  30 sec. 20 sec.                                                                             30 sec.                                                                             20 sec.                                                                             30 sec.                            ______________________________________                                         25     23.1     23.0    2.1   2.04  9.1   9.0                                 55     53.3     53.2    3.19  3.32  6.0   6.3                                101     101      101     3.0   3.3   3.0   3.3                                326     326.6    327     13.3  9.8   4.1   3.0                                501              503           17.1        3.4                                690              675           28          4.15                               810              813           37          4.5                                ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Reproducibility of a Dual Wavelength MPX System                               YSI     Average (mg/dl)                                                                            S.D. (mg/dl)                                                                              % C.V.                                       (mg/dl) 20 sec.  30 sec. 20 sec.                                                                             30 sec.                                                                             20 sec.                                                                             30 sec.                            ______________________________________                                         25      25      27      1.34  1.55  5.4   5.7                                 55      55      57.4    2.58  2.62  4.7   4.6                                101     101      101.5   2.55  2.18  2.5   2.1                                326     332      330     15.0  7.1   4.5   2.1                                501              505           21.3        4.2                                690              687           22.8        3.3                                810              817           30.4        3.7                                ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Reproducibility of a 3.0 mm Diameter Aperture                                                % C.V.                                                         YSI (mg/dl)      4.7 mm  3.0 mm                                               ______________________________________                                        55-100           4.8     4.9                                                  300              3.0     5.0                                                  600              3.8     5.5                                                  avg.             3.9     5.1                                                  ______________________________________                                    

The blood was divided into aliquots and spiked with glucose across arange of 25-800 mg/dl. Twenty determinations were made at each glucosetest level from strips taken at random from a 500 strip sample (LotFJ4-49B). The results of this study lead to the following conclusions:

1. Single vs. Dual Wavelength

The average C.V. for the 30-second dual result was 3.7% vs. 4.8% for the30-second single wavelength result, an improvement of 23% across aglucose range of 25-810 mg/dl. There was a 33% improvement in C.V. inthe 25-326 mg/dl glucose range. Here the C.V. decreased from 5.4% to3.6%, a significant improvement in the most used range. The 20-seconddual wavelength measurement gave similar improvements in C.V. comparedto the single wavelength measurement in the 25-325 mg/dl range (Tables 3and 4).

2. Dual Wavelength, 20 vs. 30-second Result

The average C.V. for a 20-second result in the 25-100 mg/dl range isnearly identical to the 30-second reading, 4.2% vs. 4.1%. However, at326 mg/dl the 30-second reading has a C.V. of 2.1% and the 20-secondresult a C.V. of 4.5%. As was seen in the K/S-20 response curve, theslope begins to decrease sharply above 250 mg/dl. This lead to poorreproducibility at glucose levels greater than 300 for the 20-secondresult. From this reproducibility data the cutoff for the 20-secondresult is somewhere between 100 and 326 mg/dl. A cutoff of 250 mg/dl waslater determined from the results of the recovery study set forth inExample II.

3. Aperture Size

A smaller optics aperture size, 3.0 mm vs. 5.0 mm, was investigated.Initial experimentation using a 10-replicate, hand-dipped disk sampledid show improved C.V.s with the 3.0 mm aperture, apparently because ofeasier registration with the system optics. However, when machine-maderoll membrane was used, the average C.V. (Table 5) of the largeraperture, 4.7 mm, was 3.9% vs. an average C.V. for the 3.0 mm apertureof 5.1%. This 30% increase in C.V. was probably due to the unevensurface of the roll membrane lot as discussed below.

EXAMPLE II Recovery

For comparison of the present method (MPX) against a typical prior artmethod using a Yellow Springs Instrument Model 23A glucose analyzermanufactured by Yellow Springs Instrument Co., Yellow Springs, Ohio(YSI), blood from 36 donors was tested. The donors were divided equallybetween males and females and ranged in hematocrit from 35 to 55%. Theblood samples were used within 30 hours of collection, with lithiumheparin as the anti-coagulant. Each blood sample was divided intoaliquots and spiked with glucose to give 152 samples in the range of0-700 mg/dl glucose. Each sample was tested in duplicate for a total of304 data points.

Response curves were constructed from these data and glucose values thencalculated from the appropriate equation (Tables 1 and 2). These MPXglucose values were then plotted vs. the YSI values to givescattergrams.

Comparison of MPX Systems: For both the 20-second and 30-secondmeasurement times there is visually more scatter in thesingle-wavelength scattergrams than the dual-wavelength scattergrams.The 20-second reading becomes very scattered above 250 mg/dl but the30-second measurement does not have wide scatter until the glucose levelis ≧500 mg/dl.

These scattergrams were quantitated by determining the deviations fromYSI at various glucose ranges. The following results were obtained.

                  TABLE 6                                                         ______________________________________                                        Accuracy of MPX from Recovery Data                                            MPX               S.D.                                                        Wave-  Measurement                                                                              (mg/dl)  C.V for Range*                                     length Time (sec.)                                                                              0-50     50-250                                                                              250-450                                                                              450-700                               ______________________________________                                        Single 20         ±5.6  7.2   14.5   --                                    Single 30         ±6.9  7.1    8.8   10.2                                  Dual   20         ±2.3  5.3   12.8   --                                    Dual   30         ±2.19 5.5    5.8    8.4                                  ______________________________________                                         Note: These are inter method C.V.s.                                           a. The dual wavelength system gave C.V.s that ranged 30% lower than the       single wavelength system.                                                     b. The single wavelength system, from 0-50 mg/dl, showed a S.D. of ±6-     mg/dl whereas the S.D. for a dual wavelength measurement was only ±2.2     mg/dl.                                                                        c. The cutoff for a 30second MPX measurement is 250 mg/dl. For the 50-250     mg/dl range both the 20 and 30second measurements gave similar intermetho     C.V.s (approximately 7% for single wavelength, 5.5% for dual wavelength).     However, in the 250-450 mg/dl range intermethod C.V.s more than double fo     the 20second reading to 14.5% for the single and 12.8% for the dual           wavelength.                                                                   d. The 30second reading was unusable above 450 mg/dl for both the single      and dual wavelength measurement (C.V.s of 10.2 and 8.4%).                

In conclusion, two MPX systems gave optimum quantitation in the 0-450mg/dl range.

1. MPX 30 Dual

This dual wavelength system gave a 30-second measurement time with a 95%confidence limit (defined as the probability of a measurement beingwithin 2 S.D. of the YSI) of 11.3% (C.V.) for the range from 50-450mg/dl (Table 7) and ±4.4 mg/dl (S.D.) for 0-50 mg/dl.

2. MPX 30/20 Dual

This dual wavelength system gave a 20-second measurement time in the0-250 mg/dl range and a 30-second time for the 250-450 range. The 95%confidence limits are nearly identical to the MPX 30 Dual System (Table7), 11.1% (C.V.) for 50-450 mg/dl and ±4.6 mg/dl (S.D. for 0-50 mg/dl).

                  TABLE 7                                                         ______________________________________                                        Comparison of 95% Confidence Limits for MPX,                                  GlucoScan Plus and Accu-Chek bG* Reagent Strips                               Measuring                                                                     Range   MPX Single Wavelength                                                                          MPX Dual Wavelength                                  mg/dl   20 sec.    30 sec.   20 sec. 30 sec.                                  ______________________________________                                        0-50    11.2 mg/dl 13.8 mg/dl                                                                               4.6 mg/dl                                                                             4.4 mg/dl                               50-250  14.4%      14.2%     10.6%   11.0%                                    250-450 --         17.6%     --      11.6%                                    77-405  GlucoScan Plus (Drexler Clinical)                                                                  15.9%                                            77-405  Accu-Chek bG (Drexler Clinical)                                                                    10.7%                                            50-450  MPX 20/30 Dual Hybrid                                                                              11.1%                                            50-450  MPX 30 Dual          11.3                                             ______________________________________                                         *Confidence limits for MPX were from the YSI. The confidence limits for       GlucoScan Plus and AccuChek bG were from the regression equation vs. YSI      which eliminates bias due to small differences in calibration.           

EXAMPLE III Stability

Most of the bench-scale work carried out in optimizing stability wascompleted using hand-dipped 0.8 μm POSIDYNE membrane disks. The specificdye/enzyme formulation was set forth previously.

1. Room Temperature (RT) Stability

This study attempted to chart any change in response of the 0.8 μmPosidyne membrane reagent stored at 18°-20° C. over silica geldesiccant. After 2.5 months there was no noticeable change as measuredby the response of a room temperature sample vs. the response of asample stored at 5° C. Each scattergram represented a glucose range of0-450 mg/dl.

2. Stability at 37° C.

A 37° C. stability study using the same reagent as the RT study wascarried out. The differences in glucose values of reagent stressed at37° C. vs. RT reagent, for strips stressed with and without adhesive,was plotted over time. Although the data was noisy, due to the poorreproducibility of handmade strips, the stability was excellent forstrips whether they were stressed with or without adhesive.

3. Stability at 56° C.

Eight 5- to 6-day stability studies were carried out using differentpreparations of a similar formulation on disk membrane (Table 8). Forthe low glucose test level (80-100 mg/dl) the average gulcose valuedropped upon stressing by 3.4%, with the highest drop being 9.55%. Atthe high test level (280-320 mg/dl) the glucose reading declined by anaverage of 3.4%, the largest decline being 10.0%.

                  TABLE 8                                                         ______________________________________                                        Stability of pH = 4.0, 0.8 μm POSIDYNE Disk Reagent                        Formulation Stressed for 5 to 6 Days at 56° C.                                 % Difference (56° C. vs. RT Sample)                            Sample No.                                                                              YSI (80-100 mg/dl)                                                                           YSI (280-320 mg/dl)                                  ______________________________________                                        FJ22B     -6.25          +5.4                                                 FJ27A     -4.0           -5.14                                                FJ28B     -2.4           -5.3                                                 FJ30H     -9.55          -10.0                                                FJ31C     +4.43          -1.24                                                FJ36      -3.2           -8.5                                                 FJ48B*    -3.0           0.0                                                  GM48A*    -3.0           -2.5                                                 Average of 8                                                                            -3.4           -3.4                                                 ______________________________________                                         *These two samples contained twice the normal concentration of enzyme and     dye.                                                                     

A study of the 56° C. stressing of this membrane over a 19-day periodshowed no major difference for strips stressed with or without adhesive.In both cases the 19-day decline in glucose value was <15% at low testlevels (80-100) and 300 mg/dl.

Another 56° C. study using hand-dipped 0.8 μm posidyne membrane withtwice the normal concentration of enzyme and dye was completed. Twoseparate preparations of the same formulation were made up and thestability measured over a 14-day period. The average results of the twostudies were plotted. Changes were within ±10% over the 14-day period atboth the high and low glucose test level. These data show thisformulation to be particularly stable.

EXAMPLE IV Sample Size

The sample size requirements for MPX are demonstrated in Table 9.

                                      TABLE 9                                     __________________________________________________________________________    Effect of Sample Size on MPX Measurements                                     Sample                                                                             Dual Wavelength  Single Wavelength                                       Size (μl)     Average          Average                                     __________________________________________________________________________    Low Glucose YSI = 56                                                          3    41 50 39 31 40   31 42 30 19 30                                          4    44 49 49 49 48   41 45 44 45 44                                          5    54 48 49 51 50   50 49 48 49 49                                          10   48 48 50 47 48   54 53 56 55 54                                          20   49 49 49 50 49   55 57 58 60 58                                          High Glucose YSI = 360                                                        3    301                                                                              260                                                                              276                                                                              286                                                                              280  274                                                                              232                                                                              244                                                                              260                                                                              252                                         4    383                                                                              378                                                                              367                                                                              341                                                                              367  361                                                                              356                                                                              342                                                                              318                                                                              344                                         5    398                                                                              402                                                                              382                                                                              370                                                                              388  378                                                                              387                                                                              366                                                                              351                                                                              370                                         10   364                                                                              362                                                                              378                                                                              368                                                                              368  356                                                                              358                                                                              379                                                                              369                                                                              366                                         20   375                                                                              370                                                                              380                                                                              378                                                                              376  380                                                                              382                                                                              389                                                                              385                                                                              384                                         __________________________________________________________________________

The volumes reported in the table were transferred to the reagent padshown in FIG. 1 using a micro pipet. When blood from a finger stick isapplied to a strip the total sample cannot be transferred, therefore thevolumes reported here do not represent the total sample size needed tobe squeezed from the finger for the analysis. A 3-μl sample is theminimum necessary to completely cover the reagent pad circle. This doesnot provide enough sample to completely saturate the reagent pad and MPXgives low results. A 4-μl sample barely saturates the reagent pad, whilea 5-μl sample is clearly adequate. A 10-μl sample is a large shiny dropand a 20-μl sample is a very large drop and is only likely to be usedwhen blood from a pipet is used for sampling.

At low glucose concentration the single wavelength result has somedependence on sample size which is completely eliminated using the dualwavelength measurement. Although this dependence with the singlewavelength might be considered acceptable, it is clearly undesirable.

EXAMPLE V Reproducibility

Experimental measurements described above were always run in replicate,usually 2, 3 or 4 determinations per data point. These sets have alwaysshown close agreement even for samples with extreme hematocrits orextreme oxygen levels. C.V.s were well below 5%. It appears, therefore,that reproducibility is very good to excellent.

The subject invention provides for many advantages over systems whichare presently available commercially or have been described in theliterature. The protocols are simple and require little technical skilland are relatively free of operator error. The assays can be carried outrapidly and use inexpensive and relatively harmless reagents, importantconsiderations for materials employed in the home. The user obtainsresults which can be understood and used in conjunction with maintenancetherapy. In addition, the reagents have long shelf lives, so that theresults obtained will be reliable for long periods of time. Theequipment is simple and reliable and substantially automatic.

All patents and other publications specifically identified in thisspecification are indicative of the level of skill of those of ordinaryskill in the art to which this invention pertains and are hereinindividually incorporated by reference to the same extent as would occurif each reference were specifically and individually incorporated byreference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many modifications and changes can bemade thereto without departing from the spirit or scope of the inventionas defined in the following claims.

What is claimed is:
 1. An apparatus for measuring reflectance from aporous matrix containing a reagent that reacts with an analyte suspectedof being present in a liquid sample applied to a surface in said porousmatrix, where said sample can migrate through said porous matrix andreact with said reagent if said analyte is present in said sample, whichcomprises:a housing; an internal chamber in said housing; means foraccessing said internal chamber, whereby said porous matrix can beinserted in or removed from said internal chamber through said means foraccessing; means for excluding external light from entering saidinternal chamber through said means for accessing; means for retainingsaid porous matrix in said internal chamber, wherein said porous matrixis retained with a first surface accessable for applying said liquidsample through said means for accessing; means for illuminating a secondsurface of said porous matrix when said porous matrix is retained insaid internal chamber, wherein said second surface is different fromsaid first surface; means for detecting intensity of light reflectedfrom said second surface of said porous matrix when said porous matrixis retained in said internal chamber and illuminated by said means forilluminating, wherein said means for detecting produces an outputrelated to said intensity, said means for detecting intensity beingadapted to provide a series of outputs; control means for processingsaid outputs, said control means including means for comparing outputsin said series and determining whether said outputs decrease by apredetermined difference sufficient to indicate that said sample of bodyfluid has reached said second surface, wherein said predetermineddecrease in outputs causes said control means to initiate a timingperiod which results in a measurement reading being taken from saidsurface at a predetermined time after said second timing period isinitiated without having determined the time at which said sample ofbody fluid was initially applied to said porous matrix; and means forreporting said measurement reading.
 2. The apparatus of claim 1, whereinsaid apparatus further comprises a self-contained electronic powersupply operationally connected to said means for illuminating, means fordetecting, control means, and means of reporting.
 3. The apparatus ofclaim 1, wherein said means for illuminating and means for detectinglight together comprise: 1)two light sources of different wavelengthsand a single light detector; or 2) a polychromatic light source and twodetectors restricted to detecting different wavelegths of light, whereinone of said wavelengths measures a product absorbance and the other ofsaid wavelengths measures a background absorbance.
 4. The apparatus ofclaim 3, wherein the first wavelength of light is from 690 to 710 nm andthe second wavelength of light is from 625 to 645 nm.
 5. The apparatusof claim 1, wherein said control means is further capable of collectingand storing a background detector current reading in the absence oflight reflected from said porous matrex.
 6. The apparatus of claim 5,further comprising a power supply, wherein said control means furthercomprises means for automatically collecting and comparing sequentialreflectance readings from said means for detecting when power issupplied to said control means in an analyte detection mode; means forinitiating a timing circuit upon detection of said decrease inreflectance; means for collecting a reaction reflectance reading at apredetermined interval after said drop in reflectance; means forcalculating a value for concentration of analyte in said liquid beinganalyzed from said reaction reflectance reading; and means fortransferring said value to said reporting means.
 7. The apparatus ofclaim 6, wherein said control means further comprise means forcollecting and storing a base reflectance reading from said matrix whenpower is supplied to said control means in a base reflectance mode priorto said analyte detection mode and means for subtracting said basereflectance reading from said reaction reflectance reading prior tocalculation by said control means of said value.